KR20020083179A - Novel LHRH antagonists, production and use thereof as medicament - Google Patents

Abstract

The present invention relates to a peptide comprising an N-methylated amino acid component and having improved water solubility. Drugs containing the peptide according to the invention may be used to treat symptoms of non-malignant diseases of hormone dependent tumors and hormone effects.

Description

[0001] The present invention relates to novel LHRH antagonists, to their preparation and to their use as medicaments.

The present invention relates to a method for the treatment of LHRH antagonists with improved solubility characteristics, methods for preparing these compounds, pharmaceuticals containing these compounds and hormone dependent tumors and hormone-affecting non-malignant disorders such as benign prostatic hyperplasia (BPH) and endometriosis To the use of medicaments.

Nomenclature used for the definition of peptides is the biochemical nomenclature [European J. Biochem. 1984, 138, 9 - 37], the amino group at the N-terminus is shown on the left and the carboxyl group at the C-terminus is shown on the right according to the convention. LH-RH antagonists such as peptides according to the present invention include naturally occurring amino acids and synthetic amino acids and natural amino acids include Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln , Cys, Met, Phe, Tyr, Pro, Trp, and His. The abbreviations for each amino acid residue are based on the common name of the three letters of the amino acid and are as follows: Ala = alanine, Arg = arginine, Gly = glycine, Leu = leucine, Lys = lysine, Phe = phenylalanine, Cpa = 4-chlorophenylalanine, Pro = proline, Ser = serine, Thr = threonine, Trp = tryptophan , Try = Tyrosine and Sar = Sarco god. All amino acids listed herein are derived from the L-series unless otherwise noted. For example, D-Nal (2) is an abbreviation for 3- (2-naphthyl) -D-alanine and Ser is an abbreviation for L-serine. Substituents for the [epsilon] -amino group of the lysine side chain are represented by the term in parentheses after the Lys, if appropriate in the form of the abbreviation.

Another abbreviation used is as follows:

Ac = acetyl,

B = 4- (4-amidinophenyl) amino-1,4-dioxobutyl,

Boc = tert-butyloxycarbonyl,

Bop = benzotriazole-1-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate,

DCC = dicyclohexylcarbodiimide,

DCM = dichloromethane,

Ddz = dimethoxyphenyl-dimethylmethyleneoxy-carbonyl (dimethoxy-dimethyl-Z),

DIC = diisopropylcarbodiimide,

DIPEA = N, N-diisopropylethylamine,

DMF = dimethylformamide,

Fmoc = fluorenylmethyloxycarbonyl,

HF = hydrofluoric acid,

HOBt = 1-hydroxybenzotriazole,

HPLC = high pressure liquid chromatography,

Me = methyl,

TFA = trifluoroacetic acid,

Z = benzyloxycarbonyl.

The peptide according to the present invention is an analogue of luteinizing-hormone-releasing hormone (LH-RH) and has the following structure:

p-Glu-His-Trp- Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2, [LH-RH, and I relrin.

For more than 20 years, researchers have sought to explore selective and potent antagonists of LH-RH decapeptides (M. Karten and J.E. Rivier, Endocrine Reviews 7, 44-66 (1986)]. These antagonists are of great interest because of their usefulness in endocrinology, gynecology, contraception and cancer. Many compounds have been prepared as potential LH-RH antagonists. The most interesting compounds discovered so far are compounds whose structure is a modification of the LH-RH structure.

A first class of potent antagonists is obtained by introducing aromatic amino acid residues at positions 1, 2, 3 and 6 or at positions 2, 3 and 6. A typical method for describing compounds is as follows: First, the amino acid occupying the position of the amino acid originally present in the peptide chain of LH-RH is indicated, and the position at which the substitution occurs is superscripted. In addition, " LH-RH " is indicated at the following positions to express that they are substituted LH-RH analogues.

Known antagonists are:

^{[Ac-D-Cpa 1,2,} D-Trp 3,6] LH-RH [ reference literature: DH Coy et al, In: . Gross, E. and Meienhofer, J. (Eds) Peptides; Proceedings of the 6th American Peptide Symposium, pp. 775-779, Pierce Chem. Co., Rockville III (1979); ^{[Ac-Pro 1, D-} Cpa 2, D-Nal (2) 3,6] LH-RH [ reference: U.S. Patent No. 4,419,347; and ^{[Ac-Pro 1, D-} Cpa 2, D-Trp 3 ^{, 6} ] LH-RH [JL Pineda, et al., J. Clin. Endocrinol. Metab. 56, 420, 1983).

Since a basic amino acid in order to improve the action of antagonists to, for example, the introduction of D-Arg in the 6-position: for ^{example, [Ac-D-Cpa 1,2} , D-Trp 3, D-Arg 6, D-Ala ¹⁰ ] LH-RH (ORG-30276) (DH Coy, et al., Endocrinology 100, 1445, 1982) ^{F) 2, D-Trp 3} , D-Arg 6] LH-RH (ORF 18260) [ reference literature: JE Rivier et al, in: . Vickery BH Nestor, Jr. JJ, Hafez, ESE (Eds). LHRH and its Analogs, pp. 11-22 MTP Press, Lancaster, UK 1984].

Further potent LH-RH antagonists are described in WO 92/19651, WO 94/19370, WO 92/17025, WO 94/14841, WO 94/13313, US-A 5,300,492, US-A 5,140,009, EP 0 413 209 A1 and DE 195 44 212 A1.

The latter describes compounds corresponding to the following formula with ornithine or lysine units modified at the 6 position:

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -Tyr ⁵ -D-Xxx ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂ (Wherein D-Xxx is an amino acid group represented by the following formula (VI)).

Further known LH-RH antagonists are Antaralix, Ganirelix, and Setorelelix.

Antaralix ^R (INN: Theverelics):

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -Tyr 5 -D-Hci 6 -Leu 7 -Lys (iPr) 8 -Pro 9 -D-Ala 10 -NH ₂

Gani Relics:

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -Tyr 5 -D-hArg (Et) 2 6 -Leu 7 -hArg (Et) 2 8 -Pro 9 -D-Ala ¹⁰ -NH ₂

Set Laurellix:

Ac-D-Nal (2) ¹ -D-Cpa ² -Pal (3) ³ -Ser ⁴ -Tyr ⁵ -D-Cit ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂

It is an object of the present invention to prepare novel LH-RH antagonists with improved enzymatic stability and significantly improved water solubility.

This object is achieved by the salts of the compounds of the formula (I) and their pharmaceutically acceptable acids, in particular acetates, embonates and trifluoroacetates.

A-Xxx ¹ -Xxx ² -Xxx ³ -Xxx ⁴ -Xxx ⁵ -Xxx ⁶ -Xxx ⁷ -Xxx ⁸ -Xxx ⁹ -Xxx ¹⁰ -NH ₂

In this formula,

A is an acetyl group,

Xxx ¹ is D-Nal (2)

Xxx ² is D-Cpa,

Xxx ³ is D-Pal (3)

Xxx ⁴ is Ser,

Xxx ⁵ is N-Me-Tyr,

Xxx ⁶ is D-Cit, D-Hci or D- [-N'- 4- (4-amidinophenyl) -amino- 1,4-dioxobutyl] Lys (abbreviation: D-Lys ego,

Xxx ⁷ is Leu or Nle,

Xxx ⁸ is Arg or Lys (iPr)

Xxx ⁹ is Pro,

Xxx ¹⁰ is D-Ala or Sar,

However, when Xxx ⁶ is D-Lys (B), Xxx ⁷ is Nle, and when Xxx ⁶ is D-Cit, Xxx ⁷ is Nle and Xxx ¹⁰ is D-Ala,

When Xxx ⁶ is D-Hci, Xxx ⁷ is Leu and Xxx ¹⁰ is D-Ala.

According to a further aspect of the invention, the following compounds and salts thereof with pharmaceutically acceptable acids are particularly preferred:

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Arg 8 -Pro 9 - Sar ¹⁰ -NH ₂ ,

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Arg 8 -Pro 9 - D-Ala ¹⁰ -NH ₂ ,

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Lys (iPr) 8 - Pro ⁹ -Sar ¹⁰ -NH _2,

Ac-D-Nal (2) 1 -D-Phe (4-Cl) 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Lys (iPr) ^8- Pro ^9- D-Ala ¹⁰ -NH ₂ ,

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Cit ⁶ -Nle ⁷ -Arg ⁸ -Pro ⁹ -D- ¹⁰ -NH ₂ ,

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ^6- Leu ⁷ -Arg ⁸ -Pro ⁹ -D- ¹⁰ -NH ₂ ,

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 -Lys (iPr) 8 -Pro 9 - D-Ala ¹⁰ -NH ₂ and

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Hci 6 -Leu 7 -Lys (iPr) 8 -Pro 9 - D-Ala ¹⁰ -NH ₂ .

According to a further aspect of the present invention, the compounds of the present invention are present as acetate, trifluoroacetate or embonate salts.

According to a further aspect of the present invention, the compounds of the present invention can be used as drugs or medicaments.

According to a further aspect of the present invention there is provided a medicament containing one or more compounds according to the invention, conventional vehicles and excipients.

According to a further aspect of the present invention, fragments from Xxx ^m where m is an integer from 1 to 10 and Xxx ¹ is acetylated, provided with a suitable protecting group are synthesized in solid phase or in solution according to conventional methods Followed by coupling of the fragment to the solid phase by means of fragment coupling followed by coupling and subsequent removal of the compound of formula I from the solid phase using conventional methods while amidating the Xxx ¹⁰ unit. A method for manufacturing the same is provided. According to a further aspect of the present invention there is provided the use of a compound of the invention for the manufacture of a medicament for the treatment of hormone dependent tumors, in particular prostate cancer or breast cancer, and non-malignant syndrome requiring the inhibition of LH-RH hormone / RTI > According to a further aspect of the present invention there is provided a process for the preparation of a medicament which comprises mixing at least one compound according to any one of claims 1 to 10 with a conventional vehicle and excipient and formulating it as a medicament. In accordance with a further aspect of the present invention there is provided a method of treating or preventing a hormone dependent tumor, particularly prostate cancer, breast cancer or uterine leiomyoma, in a mammal, particularly a human, by administering an effective amount of at least one compound according to the present invention, A method for treating non-malignant symptoms, for example, endometriosis, benign prostatic hyperplasia (BPH) and male or female infertility, which need to be inhibited.

The compounds according to the present invention are useful for the treatment and prophylaxis of hormone dependent tumors, in particular prostate cancer, breast cancer or uterine myoma, and non-malignant symptoms which need to inhibit LH-RH hormone for treatment, for example endometriosis, benign prostatic hyperplasia ). ≪ / RTI > Additionally, they can be used to treat male or female infertility by, for example, modulating the ovarian hyperstimulation during the in vitro fertilization process. For this purpose, they are usually mixed with conventional vehicles and excipients and formulated as medicaments in a known manner. The compounds according to formula (I) are conventional fragment condensation or by using the already acylated D- lysine in the side chain to the carboxylic acid of formula R ¹ -COOH, or deca-peptide monomer and the carboxylic acid reaction by a suitable amide bond in the side chain of D- lysine ⁶ And then synthesizing them by sequential synthesis. Thus, the R ¹ -CO group can be introduced into the peptide chain in three different steps of the process, i. E., After lysine or ornithine is introduced into the peptide chain before condensation of the respective unit to obtain the peptide, All or all of the monomers may be introduced after condensation. Compounds of formula I may be prepared by known methods, for example, by the use of pure solid phase techniques, partial solid phase techniques (so-called fractional condensation) or conventional solution coupling methods (M. Bodanszky, " Principles of Peptide Synthesis & Verlag 1984]. For example, the solid phase synthesis method is described in textbook [" Solid Phase Peptide Synthesis ", JM Stewart and JD Young, Pierce Chem. Company, Rockfield, III, 1984 and in G. Barany and RB Merrifield " The Peptides ", Ch. 1, pp. 1-285, 1979, Academic Press Inc.]. Typical solution synthesis methods are described in Houben-Weyl, Synthese von Peptiden, E. Wunsch (Editor) 1974, Georg Thieme Verlag, Stuttgart, FRG]. For example, first, a carboxy-terminal amino acid protected with an? -Amino group is first bound to an insoluble support and the? -Amino protective group of the amino acid is removed, and then the obtained free amino Linking the group to the carboxyl group of the next protected amino acid so that the conventional amino acids of the peptide are ligated to be synthesized in a step-wise and precise sequence, removing the final peptide from the support after all amino acids are connected, Stepwise synthesis is performed to remove the protecting group. Stepwise condensation is typically carried out in a protected Is synthesized from the corresponding amino acid in a conventional manner.

Each amino acid is linked to one another according to conventional methods and suitable methods are as follows:

A symmetrical anhydride process in the presence of dicyclohexylcarbodiimide or diisopropylcarbodiimide (DCC, DIC)

- General carbodiimide method,

Carbodiimide / hydroxybenzotriazole method [The Peptides, Volume 2, Ed. E. Gross and J. Meienhofer]

In fragment coupling, azide coupling methods without racemization, DCC-1-hydroxybenzotriazole or DCC-3-hydroxy-4-oxo-3,4-dihydro- The benzotriazine method is preferably used. Activated esters of the fragment may also be used.

Esters of N-protected amino acids, such as N-hydroxysuccinimide esters or 2,4,5-trichlorophenyl esters, are particularly well suited for step-wise condensation of amino acids. Amino hydrolysis can be very well catalyzed by an N-hydroxy compound having an acidity of approximately acetic acid, for example, 1-hydroxybenzotriazole.

The present intermediate amino protecting group itself is a group which can be removed by hydrogenation, for example a benzyloxycarbonyl radical (= Z radical) or a weak acid. Suitable protecting groups for the a-amino group include, for example, tertiary butyloxycarbonyl groups, fluorenylmethyl-oxycarbonyl groups, carbobenzoxy groups or carbobenzothio groups (in each case optionally p-bromo - [as described above] or a p-nitrobenzyl radical), a trifluoroacetyl group, a phthalyl radical, an o-nitrophenoxyacetyl group, a trityl group, a p-toluenesulfonyl group, Benzyl radicals substituted in the nucleus (p-bromo- or p-nitrobenzyl radicals) and [alpha] -phenylethyl radicals. The references cited herein are as follows: P. Greenstein and Milton Winitz, Chemistry of Amino Acids, New York 1961, John Wiley and Sons, Inc., Volume 2, for example page 883 et seq., "Principles of Peptide Synthesis ", Springer Verlag 1984, " Solid Phase Peptide Synthesis ", JM Stewart and JD Young, Pierce Chem. Company, Rockford, III, 1984, G. Barany and RB Merrifield " The Peptides ", Ch. 1, pp. 1-285, 1979, Academic Press Inc., and also ThePeptides, Volume 2, Ed. E. Gross and J. Maienhofer, Academic Press, New York. These protecting groups are also basically suitable for the protection of additional functional side groups (OH groups, NH ₂ groups) of the corresponding amino acids.

The hydroxyl groups present (serine, threonine) are preferably protected by a benzyl group and similar groups. Additional amino groups that are not at the? -position (e.g., the amino group at the? -position, the guanidino group of arginine) are preferably protected at a right angle.

Except for the lysine modified by the R ¹ -CO- group [abbreviated], each amino acid unit can be commercially obtained.

Possible methods for preparing the lysine modified by the R < ^{1 >} -CO- group are as follows:

1. The? -Carboxylic acid groups are suitably protected, for example, by esterification.

2. The ε-amino group is protected, for example, by the Z group.

3. The? -Amino group can be protected, for example, with a Boc group to selectively remove the amino protecting group.

4. Remove the Z group on the epsilon-amino group.

5. Introduce the desired group R < ^{1 >} -CO- on the [epsilon] -amino group.

6. Remove the protecting group on the [alpha] -amino group.

7. The alpha -amino group is reversibly derivatized optionally using, for example, the Z group.

A suitable method for reacting the amino group of a lysine with an appropriate carboxylic acid or carboxylic acid derivative to introduce the R < ^{1 >} -CO- group is basically the same as the method for coupling an amino acid as described above. However, it is particularly preferred to condense using carbodiimides, for example 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole.

The reaction for coupling the amino acid can be carried out in a conventional inert solvent (e.g. dichloromethane) or suspension and optionally dimethylformamide can be added to improve the solubility.

A suitable synthetic support is an insoluble polymer which can swell in an organic solvent (e.g., a copolymer of polystyrene and 1% divinylbenzene), for example a polystyrene resin in bead form. The protected decapeptide amide on the methylbenzhydrylamine resin (MBHA resin, i.e., the polystyrene resin provided with the methylbenzhydrylamine group) that provides the desired C-terminal amide functionality of the peptide after HF cleavage from the support They can be synthesized according to the steps in the table:

table

Peptide synthesis protocol using Boc-protected amino acids

step Action Solvent / reagent (v / v) time One wash Methanol 2 x 2 min 2 wash DCM 3 x 3 min 3 remove DCM / TFA (1: 1) 1 x 30 min 4 wash Isopropanol 2 x 2 min 5 wash Methanol 2 x 2 min 6 wash DCM 2 x 3 min 7 Neutralization DCM / DIPEA (9: 1) 3 x 5 min 8 wash Methanol 2 x 2 min 9 wash DCM 3 x 3 min 10 End Addition of Boc-As in DCM + DIC + HOBt 11 Coupling DCM, optionally DCM / DCF About 90 minutes 12 wash Methanol 3 x 2 min 13 wash DCM 2 x 3 min

The N [alpha] -Boc-protected amino acid was coupled with a 3 fold molar excess in the presence of diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in CH ₂ Cl ₂ / 50% trifluoroacetic acid (TFA) in CH ₂ Cl _{2 is added} to remove the Boc-protecting group.

To ensure complete conversion, a chloranil test can be used according to Christensen and Kaiser's Ninhydrin test. The radical of the liberated amino functional group is blocked by acetylation with acetylimidazole in a 5-fold excess in CH ₂ Cl ₂ . Peptides are synthesized on the dendrites following a series of reaction steps in Table 1. To remove the resin bound peptide, each final product of the solid phase synthesis is vacuum dried with P ₂ O ₅ and treated at 0 ° C for 60 min at 500: 1 excess of HF / anisole 10: 1 / v: v .

HF and anisole under vacuum is distilled off and washed with anhydrous ethyl ether with stirring to obtain the peptide amide as a white solid and the additionally obtained polymeric support is washed off with a 50% strength acetic acid solution. By carefully concentrating the acetic acid solution in vacuo, each peptide can be obtained as a highly viscous oil which is converted to a white solid after addition of ether in the cold solution.

And further purified using conventional methods for high pressure liquid chromatography (HPLC).

The peptide can be reacted with an acid to convert it into its acid addition salt in a known manner. Conversely, an acid addition salt thereof can be reacted with a base to give a free peptide. The peptide embonate can be prepared by reacting the trifluoroacetic acid salt (TFA salt) of the peptide with the free disodium salt of embornic acid (pamoic acid) or embic acid. To this end, the peptide TFA salt is treated with disodium embonate in a polar aprotic medium, preferably dimethylacetamide, in aqueous solution and the resulting pale yellow precipitate is isolated.

The following examples are provided to illustrate the present invention but are not intended to be limiting thereof.

Example 1 (D-68968)

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Arg 8 -Pro 9 - Sar ¹⁰ -NH ₂

The decapeptide is synthesized on a polymeric support (aminomethyl-substituted resin, Fmoc protection, D-1675 form, Bachem) with a loading density of 0.55 mmol / g. The lysine is coupled as Fmoc-D-Lys (Boc) -OH and the Fmoc protecting group is removed using 20% piperidine / DMF. All the side-chain protecting groups are removed at the same time and the separated peptides are cleaved from the polymeric support and purified using preparative HPLC. After freeze-drying, a 98.5% concentration decapeptide is obtained.

The nitrogen of D-lysine is replaced with 4- (4-aminophenyl) amino-1,4-dioxobutyric acid using PyBop in DMF with addition of DIPEA. The separated crude peptide is purified using a pre-HPLC. Followed by lyophilization to give a product (trifluoroacetate) at approximately 99% concentration of the empirical C ₈₂ H ₁₀₆ ClN ₁₉ O ₁₅ with the correct FAB-MS 1633 (M + H) (calculated 1631.78096).

Example 2 (D-68969)

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Arg 8 -Pro 9 - D-Ala ¹⁰ -NH ₂

The decapeptide is synthesized on a polymeric support (aminomethyl-substituted resin, Fmoc protection, D-1675 form, Bachem) with a loading density of 0.55 mmol / g. The lysine is coupled as Fmoc-D-Lys (Boc) -OH and the Fmoc protecting group is removed using 20% piperidine / DMF. All of the side-chain protecting groups are removed at the same time and the polymerized support is removed and the isolated crude peptide, about 71% (HPLC) in content, is reacted without further purification.

The side chain of D-lysine is replaced with 4- (4-aminophenyl) amino-1,4-dioxobutyric acid using PyBop in DMF with addition of DIPEA. The separated crude peptide is purified using a pre-HPLC. Followed by lyophilization to give a product (trifluoroacetate) at approximately 98.8% concentration of the empirical C ₈₂ H ₁₀₆ ClN ₁₉ O ₁₅ with the correct FAB-MS 1633 (M + H) (calculated 1631.78096).

Example 3 (D-68971)

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Lys (iPr) 8 - Pro ⁹ -Sar ¹⁰ -NH ₂

The decapeptide is synthesized on a polymeric support (aminomethyl-substituted resin, Fmoc protection, D-1675 form, Bachem) with a loading density of 0.55 mmol / g. The lysine is coupled as Fmoc-D-Lys (Boc) -OH and the Fmoc protecting group is removed using 20% piperidine / DMF. All the side-chain protecting groups are simultaneously removed and the polymerized support is desorbed to separate the crude peptide at a content of about 59% (HPLC) using purified HPLC. After lyophilization, a 95% concentration of decapeptide is obtained.

The side chain of D-lysine is replaced with 4- (4-aminophenyl) amino-1,4-dioxobutyric acid using PyBop in DMF with addition of DIPEA. The separated crude peptide is purified using a pre-HPLC. This is followed by lyophilization to give a product (trifluoroacetate) at about 96.6% concentration of the empirical C ₈₅ H ₁₁₂ ClN ₁₇ O ₁₅ with the correct FAB-MS 1648 (M + H) (calculated 1645.8218).

Example 4 (D-68987)

Ac-D-Nal (2) 1 -D-Phe (4-Cl) 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Lys (iPr) ^8- Pro ^9- D-Ala ¹⁰ -NH ₂

D-Lys-6-unsubstituted decapeptide is synthesized on 9.09 g of a polymeric support having a loading density of 0.55 mmol / g and lysine ⁶ is coupled as Fmoc-D-Lys (Boc) -OH.

8.15 g of the crude peptide is isolated after removal from the resin. The crude peptide is purified by preparative HPLC. The side chain of D-lysine is replaced with 4- (4-aminophenyl) amino-1,4-dioxobutyric acid using PyBop in DMF with addition of DIPEA. The separated crude peptide is purified using a pre-HPLC. The product (trifluoroacetate) at about 94.6% concentration of the empirical C ₈₅ H ₁₁₂ ClN ₁₇ O _{15 with} the corresponding FAB-MS 1646.8 (M + H) (calculated 1645.82) is then obtained after lyophilization.

Example 5

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Cit ⁶ -Nle ⁷ -Arg ⁸ -Pro ⁹ -D- ¹⁰ -NH ₂

Example 6:

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ^6- Leu ⁷ -Arg ⁸ -Pro ⁹ -D- ¹⁰ -NH ₂

Example 7

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 -Lys (iPr) 8 -Pro 9 - D-Ala ¹⁰ -NH ₂

Example 8

Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Hci 6 -Leu 7 -Lys (iPr) 8 -Pro 9 - D-Ala ¹⁰ -NH ₂

General post-treatment steps for preparing the peptides according to Examples 5 to 8

Decapeptides can be prepared by both Merrifield solid phase synthesis method (SPPS) [as described above] and conventional fractional condensation methods in solution. Sequential synthesis of peptides on a polymeric support is preferred for economic reasons and can be synthesized in principle according to (1) Boc or (2) Fmoc method. Correspondingly, in each case the methylbenzhydrylamine resin (for 1) or the Fmoc-2,4-dimethoxy-4 '- (carboxymethyloxy) benzhydrylamine resin (for 2) It can be used for C-terminal bonding.

Solid phase synthesis, Merrifield method

The decapeptide was polymerized on a polymeric support Fmoc-2,4-dimethoxy-4 '- (carboxy-4'-deoxycholate) with a loading density of about 0.55 mmol / g and a particle size of 200-400 mesh under standard reaction conditions for solid phase synthesis Methyloxy) benzhydrylamine resin (Bachem D1675).

The synthesis is carried out in a sequential step on the resin phase using the N.Fmoc-protected amino acid according to the steps of Table I below.

step Action menstruum time Number of repetitions One wash DMF 2 minutes 2 x 2 remove A solution of 20% piperidine in DMF 5 minutes 2 x 3 wash DMF 2 minutes 2 x 4 wash Isopropanol 2 minutes 1 x 5 wash DMF 2 minutes 2 x 6 wash Isopropanol 2 minutes 1 x 7 wash DMF 2 minutes 2 x 8 Coupling Boc-AS-OH, HOBT, DIC in DMF 90 minutes 1 x 9 wash DMF 2 minutes 1 x 10 wash Isopropanol 2 minutes 1 x 11 wash DMF 2 minutes 1 x 12 wash Isopropanol 2 minutes 1 x 13 wash DMF 2 minutes 1 x 14 wash Isopropanol 2 minutes 1 x 15 Confirm Chloranil color test ^* 2 minutes 1 x ^* T. Christensen, Acta Chem. Scand. B 33, 763-766, 1979]

In the solid phase synthesis of decapeptides according to the steps of Table I above, the typical (repetitive) response coefficient

- Removal of the Fmoc protecting group using 20% piperidine in DMF for 2 x 5 min at room temperature (step 2).

(Step 8) with 3-fold molar excess of Fmoc amino acid using diisopropylcarbodiimide (DIC) in hydroxybenzotriazole (HoBt).

Removal of the C-terminus from the polymeric support comprising removing the amino acid side chain protecting group using trifluoroacetic acid (TFA).

When 5 g of the resin is used after removal from the polymerization support, about 5 to 6 g of the crude peptide mixture having a content of the desired component of about 70 to 80% is formed. This is recovered by subsequent preparative HPLC chromatography.

Purification of decapeptide by HPLC

Chromatographic conditions

Dynamax column RP18, 12 mu m, 300 ANGSTROM, L = 250 mm, ID = 41.4 mm,

40% B & 90% B, 50 minutes,

Solvent A: H ₂ O 970 ml + CH ₃ CN 30 ml + CF ₃ COOH 1 ml,

Solvent B: H ₂ O 300 ml + CH ₃ CN 700 ml + CF ₃ COOH 1 ml,

UV detection, = 220 nm, flow rate 60 ml / min.

The resulting fraction is concentrated in vacuo and lyophilized. Decapeptide is formed as a light colorless substance.

Followed by two cleavages in the form of the desired acetate salt for pharmacological development using chromatographic ion exchange.

Investigation of physiological function

The compounds according to formula I of the present invention are investigated for their receptor binding. The method is described in Beckers et al., Eur. J. Biochem. 231, 535-543 (1995). The setroellexes obtained according to the above synthesis were synthesized using [ ¹²⁵ I] (Amersham; Germany) using IodoGen reagent (Pierce). Inert 80.5 Bq / fmol]. The reaction mixture is purified by reverse phase high performance liquid chromatography to obtain a single iodinated set Lorelix without unlabeled peptide. In each case, about 80% of [ ¹²⁵ I] -set Lorelix and the unlabeled compound of the present invention are suitable for specific receptor binding.

The compounds of the present invention can be tested for their in vitro action using the following methods 1 and 2: The binding affinity in the binding assay of Method 1 is measured with [ ¹²⁵ I] -set Laurelix and the efficacy Tryptorelin as a stimulant is used to measure the agonistic activity.

Method 1 (measurement of K _D using set Laurellix)

Selection and characterization of mammalian cell lines with stable overexpression of human pituitary receptors for gonadoliberin (GnRH) " Eur. ≪ RTI ID = 0.0 > J. Biochem. 231, 535-543].

To investigate receptor binding, the setroellex is iodinated with [ ¹²⁵ I] (Amersham; 80.5 Bq / fmol inert) using iodogen reagent (Pierce). The high performance liquid chromatography is purified by replacing the reaction mixture with the phase and the monoiodinated set Lorelix is obtained without the unlabeled peptide. About 80% of the [ ¹²⁵ I] set Laurelix can specifically bind to the receptor.

Receptor binding assays are carried out under physiological conditions using intact cells as described in the literature (Beckers et al., 1995). Subcontent cultures of LTK ^- cells expressing stably transfected human LHRH receptors were incubated with NaCl / P _i (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO ₄ , 11.47 mM KH ₂ PO ₄ ) / 1 mM Separate by centrifugation in EDTA, centrifuge and collect. The cell pellet was resuspended in binding buffer [DMEM without H ₂ CO ₃ , 4.5 g / l glucose, 10 mM HEPES pH 7.5, 0.5% (mass / volume) BSA, 1 g / l bacitracin, 0.1 g / Mass / volume) containing NaN ₃ ]. For displacement analysis, 0.25 x 10 ⁶ cells per 100 μl were mixed with about 225 pM of [ ¹²⁵ I] -set lorelix (inactive 5 to 10 x 10 ⁵ dpm / pmol) and various concentrations of the unlabeled compound of the invention Lt; / RTI > The cell suspension in 100 [mu] l of the binding medium is laminated in 400 [mu] l assay tubes for 200 [mu] l of 84 vol% silicon oil (Merck Type 550) / 16 paraffin oil. After incubation for 1 hour at 37 ° C with gentle shaking, the cells are separated from the incubation medium by centrifugation (rotor type HTA13.8; Heraeus Sepatec, Osterode / Germany) at 9000 rpm for 2 minutes. Cut the edge of the tube containing the cell pellet. The cell pellet and supernatant are then analyzed by counting gamma radiation. The amount of non-specifically bound material is measured at a final concentration of 1 [mu] M by the addition of unlabeled set Laurellix and the total bound material is typically 10% or less. The binding data are analyzed using an EBDA / ligand analysis program (Biosoft V3.0).

The K _D value of set Laurellix is 170 picomol (pM) per liter (number of runs performed independently: 21).

Method 2 (action assay for determining antagonistic activity (IC ₅₀ value))

Reilander, H., Hilgard, P. (1997) " Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay ", Analyt. Biochem. 251, 17-23 (Beckers et al., 1997). 10,000 cells per well expressing the human LHRH receptor and the luciferase receptor gene are incubated for 24 hours in microtiter plates with DMEM together with additive and 1% (v: v) FCS _i . Then, the cells for 6 hours, thus stimulated using ^{1nM [D-Trp 6] LHRH} . Antagonistic compounds of the invention are added prior to stimulation and the cells are lysed at the end to quantify cellular Luc activity. IC ₅₀ values were calculated from the effective dose curves using the heel model [Mr. Grunwald's program EDX 2.0, Arzneimittelwerk Dresden].

Luc activity is quantified essentially twice as described in the literature (see Promega Technical Bulletins # 101/161) using the respective luciferase assay system (Promega E4030). Luciferyl-CoA is oxidized with favorable kinetics by adding coenzyme A (CoA). (Tris-phosphate 25 mM pH 7.8, dithiothreitol 2 mM, 1, 2-diaminocyclohexane-N, N, N ', N'-tetraacetic acid (CDTA)) after removal of the culture medium from the microtiter plate. (V: v), 1% (v: v) of Triton X-100) was added to the solution and dissolved. After incubation at room temperature for 15 minutes, 10 세포 of cell lysate is transferred to a suitable white microtiter plate (Dynatech) for luminescence detection. The assay buffer (Tricine 20 mM pH 7.8, (MgCO ₃ ) ₄ Mg (OH) ₂ 1.07 mM, MgSO ₄ 2.67 mM, Ethylenediaminetetraacetic acid (EDTA) 0.1 mM, Dithiothreitol 33.3 mM, Coenzyme A 270 μM, Glow 50 μl of glow-worm luciferin 470 μM, rATPNa ₂ 530 μM) is added to initiate the enzyme reaction. After 1 minute, light emission with a signal half-life of 5 minutes is measured for 1 second using EG & G Berthold MicroLumat LB 96P.

Physiochemical data and in vitro data of the compounds of the present invention are summarized in Table 2. IC ₅₀ represents a functional activity and pM represents picomoles per liter. The water solubility is measured according to the method described in Note 2).

compound Water solubility ² [mg / ml] IC ₅₀ [pM] Set Laurellix 0.002 198 (5) ¹ Example 1 (D-68968) 1.03 1300 (1) ¹ Example 2 (D-68969) 1.11 1400 (1) ¹ Example 3 (D-68971) 1.36 4700 (1) ¹ Example 4 (D-68987) 1.18 700 (1) ¹ Note 1) Numbers in parentheses indicate the number of independent experiments. 2) The water solubility is measured according to the method described below.

Solubility according to the official gadget method in Ringer's solution

To measure the solubility according to the official gadget method, the test material is mixed with an inert carrier material such as, for example, sand in excess and filled into a glass column (capacity size about 10 ml). A sieve consisting of cotton wool and a glass fiber filter is inserted into the bottom of the column in advance. The material-sand mixture is swollen for 1 hour in 1.0 ml of solvent in which the solubility is measured. Then, 10 ml of the solvent is poured into a glass column. The solution is circulated using a peristaltic pump. Measure at room temperature (about 20 ° C). The solubility is measured when the concentration of the continuous fraction is constant. The mass concentration is determined using the HPLC method described below as described above.

HPLC method:

Device

HPLC system: Hewlett Packard 1100; Detector: Hewlett Packard DAD Series 1100

Column: Column Material: Nucleosil ^R 120-3 C ₁₈

Particle size: 3㎛

Column size: 125 x 4mm

Manufacturer: Macherey & Nagel

Unit count: Injection volume: 15 μl

Flow rate: 1.0 ml / min

Oven temperature: 45 ° C

Wavelength: 226 nm

End time: 15 minutes

55% Mobile phase A: 970 ml of Milli-Q-H2O, 30 ml of acetonitrile and 1 ml of trifluoroacetic acid are mixed. The pH obtained is about 1.9.

45% mobile phase B: 300 ml of Milli-Q-H2O, 700 ml of acetonitrile and 1 ml of trifluoroacetic acid are mixed. The pH obtained is about 1.8.

The compounds of the present invention may be administered in different forms suitable for peptide active compounds. Methods well known to those skilled in the art are suitable. For example, by injection. For example, it can be administered parenterally. Subcutaneous (sc), intramuscular (i.m.), intravenous (i.v.), buccal (e.g. intraoral) or rectal administration are preferred. Intrathecal and intramuscular administration is particularly preferred. The compounds of the present invention are suitable for the preparation of different dosage forms (e. G., Lyophilizates, solutions or suspensions). Suitable dosage forms and methods for their manufacture are well known to those skilled in the art. Suitable excipients and bulk formulations include, for example, hexitol, for example mannitol, in particular D-mannitol, L-mannitol or D, L- mannitol, sorbitol such as D- sorbitol, D- or L- Alditol, iditol, glucitol and dulcitol. Are prepared according to known methods, for example, by mixing, suspending or lyophilizing methods.

Example A: A lyophilized product for the preparation of subcutaneous injection solutions

1 mg of the compound according to Example 1 (corresponding to 0.26 to 0.27 mg of acetate salt); 0 to 16.9 parts by weight of D-mannitol, preferably in each case 0.1 to 7 parts by weight, based on Example 1, and water for injection (for preparing injection solutions from lyophilisates).

Preparation: 1.62 g of the compound of Example 1 is dissolved in 30% strength acetic acid (about 1.5 L of water for injection and 91.17 g of acetic acid). Dilute the solution with 1.5 L water. 82.2 g of mannitol is added via a sterile filter and dispensed into sterile 2 ml sterile vials under sterile conditions and lyophilized. 1 mg of lyophilisate of the compound of Example 1 is obtained.

The compounds of the invention are suitable for the treatment of, for example, malignant or non-malignant hormone-dependent disorders such as breast cancer, prostate cancer, endometriosis, uterine myoma, benign prostatic hyperplasia (BPH) and male or female infertility It is also suitable, for example, for preventing immature ovulation in a patient undergoing controlled ovarian stimulation with ovarian cessation and reproductive assistance techniques. The treatment as described above may be carried out in mammals, especially humans.

Claims (16)

A salt of a compound of formula I and its pharmaceutically acceptable acid. Formula I A-Xxx ¹ -Xxx ² -Xxx ³ -Xxx ⁴ -Xxx ⁵ -Xxx ⁶ -Xxx ⁷ -Xxx ⁸ -Xxx ⁹ -Xxx ¹⁰ -NH ₂ In this formula, A is an acetyl group, Xxx ¹ is D-Nal (2) Xxx ² is D-Cpa, Xxx ³ is D-Pal (3) Xxx ⁴ is Ser, Xxx ⁵ is N-Me-Tyr, Xxx ⁶ is D-Cit, D-Hci or D- [-N'- 4- (4-amidinophenyl) -amino- 1,4-dioxobutyl] Lys (abbreviation: D-Lys ego, Xxx ⁷ is Leu or Nle, Xxx ⁸ is Arg or Lys (iPr) Xxx ⁹ is Pro, Xxx ¹⁰ is D-Ala or Sar, When Xxx ⁶ is D-Lys (B), when Xxx ⁷ is Nle and Xxx ⁶ is D-Cit, Xxx ⁷ is Nle and Xxx ¹⁰ is D-Ala, When Xxx ⁶ is D-Hci, Xxx ⁷ is Leu and Xxx ¹⁰ is D-Ala. The method of claim 1 wherein the formula Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Arg ⁸ -Pro ⁹ -Sar ¹⁰ -NH ₂ , or a pharmaceutically acceptable acid addition salt thereof. The method of claim 1 wherein the formula Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂ or a salt thereof with a pharmaceutically acceptable acid. The method of claim 1 wherein the formula Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B) 6 -Nle 7 -Lys (iPr) ^8- Pro ^9- Sars ¹⁰ -NH ₂ , or a pharmaceutically acceptable acid addition salt thereof. The method of claim 1 wherein the formula Ac-D-Nal (2) 1 -D-Phe (4-Cl) 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Lys (B ) ^6- Nle ⁷ -Lys (iPr) ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂ , or a pharmaceutically acceptable acid addition salt thereof. The method of claim 1 wherein the formula Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 -Arg 8 -Pro ^9- D-Ala ¹⁰ -NH ₂ or a salt thereof with a pharmaceutically acceptable acid. The method according to claim 1, wherein the compound represented by the formula Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Leu ⁷ -Arg ⁸ -Pro ^9- D-Ala ¹⁰ -NH ₂ or a salt thereof with a pharmaceutically acceptable acid. According to claim 1, wherein the formula Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Cit 6 -Nle 7 -Lys ( iPr) ^8- Pro ^9- D-Ala ¹⁰ -NH ₂ or a pharmaceutically acceptable acid. According to claim 1, wherein the formula Ac-D-Nal (2) 1 -D-Cpa 2 -D-Pal (3) 3 -Ser 4 -N-Me-Tyr 5 -D-Hci 6 -Leu 7 -Lys ( iPr) ^8- Pro ^9- D-Ala ¹⁰ -NH ₂ or a pharmaceutically acceptable acid. 10. Compounds according to any one of claims 1 to 9, wherein the salt is acetate, trifluoroacetate or embonate. 11. A compound according to any one of claims 1 to 10 for use as a medicament. 10. A medicament comprising at least one compound according to any one of claims 1 to 10 and a conventional vehicle and excipient. Fragments from the Xxx ^m (where m is an integer from 1 to 10 and the Xxx ¹ is acetylated) units provided with a suitable protecting group are synthesized according to conventional methods in solid or solution, and the fragments are then isolated by intercept coupling A compound of formula I according to any one of claims 1 to 9, which comprises coupling to a solid phase and coupling and then removing the compound of formula I from the solid phase using conventional methods of amidation of the Xxx ¹⁰ unit ≪ / RTI > The present invention relates to the use of the compounds of formula I in the treatment of hormone dependent tumors, especially prostate cancer, breast cancer or uterine myoma, and non-malignant symptoms which require the inhibition of LH-RH hormone for treatment, such as endometriosis, benign prostatic hyperplasia ) Or the use of a compound according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of male or female infertility. 12. A method of preparing a medicament according to claim 12, comprising mixing at least one compound according to any one of claims 1 to 10 with a conventional vehicle and excipient and formulating it as a medicament. 10. A method of treating a mammal, particularly a human, by administering an effective amount of at least one compound according to any one of claims 1 to 10 to a mammal, particularly a human, for the treatment of hormone dependent tumors, particularly prostate cancer, breast or uterine myoma, (E. G., Endometriosis, benign prostatic hyperplasia (BPH), and male or female infertility).